Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4396-4404.doi: 10.3864/j.issn.0578-1752.2021.20.013

• HORTICULTURE • Previous Articles     Next Articles

Screening and Sequence Analysis of BAC Clone Contained PG Gene Controlling Clingstone/Freestone Characteristic of Peach

MENG JunRen(),NIU Liang,DENG Li,PAN Lei,LU ZhenHua,CUI GuoChao,WANG ZhiQiang(),ZENG WenFang()   

  1. Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences/National Peach and Grape Improvement Center/Key Laboratory of Fruit Breeding Technology of Ministry of Agriculture, Zhengzhou 450009
  • Received:2020-12-03 Accepted:2021-02-26 Online:2021-10-16 Published:2021-10-25
  • Contact: ZhiQiang WANG,WenFang ZENG E-mail:82101186035@caas.cn;wangzhiqiang@caas.cn;zengwenfang@caas.cn

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Abstract:

【Objective】Tandem repeats and large segment deletions of Polygalacturonase (PG) gene was related to the clingstone/freestone characteristic of peach (Prunus persica) fruit. In this study, the sequence character of F-M locus of clingstone peach was analyzed to provide a basis for the development of related molecular markers. 【Method】With a Bacterial Artificial Chromosome library of clingstone peach 87-7-1 constructed, the positive BAC clone contained F-M locus was screened from the BAC library by PCR analysis. The screened BAC clone was sequenced by single-molecule nanopore technology. Gene annotation and sequence alignment were performed by bioinformatics. 【Result】PCR primers were designed based on re-sequencing data of existing peach varieties, and PCR reactions were performed with all the BAC library clones. Amplification products of the sequences in upstream/downstream of F-M loci were corrected, and then the target clone 46-B-10 was obtained. Full-length sequencing showed that the fragment with the length of 111612 bp and GC content of 37.03% was inserted between upstream and downstream primers. The homologous region of 46-B-10 was determined by sequence alignment with the reference genome Prunus_persica_v2.0. Five genes (Prupe.4G261700, Prupe.4G261800, Prupe.4G261900, Prupe.4G262000, and Prupe.4G262500) in F-M locus were found in the BAC clone 46-B-10. In comparison, 87-7-1 with 34 kb sequence including four genes was discarded, and one of them was EndoPGF, which controlled peach freestone and was reported previously. 【Conclusion】Compared with the reference genome freestone variety ‘Lovell’, there was only EndoPGM (Prupe.4G261900) in F-M locus of clingstone peach individual 87-7-1, while EndoPGF (Prupe.4G262200) was discarded. In this study, the structural variation of F-M locus in clingstone peach was determined, which has laid an important foundation for the development of molecular markers for clingstone/freestone trait in peach.

Key words: peach, clingstone/freestone, PG gene, BAC library

Table 1

Upstream and downstream primers of F-M loci and amplification sequences"

位置
Location		 引物名称
Primer code		 引物序列
Primer sequence		 片段大小
Size ( bp)		 扩增序列
Amplification sequence
F-M基因座上游
Upstream of F-M		 8771-PG F1 CACTAATGCTATGCGATTGTGA 178 CACTAATGCTATGCGATTGTGATCTTAACATGTAAAAATTTGCGATTAATACCTATTACAGAGAAGACTCTGCTAGATTATTGATAGATCCCTCCTTGTTTGTCCAAGTAGGGATTAAGGATAGTTTAAGTAGTGGGCTCACTTCTCTCGAGTTTATCCAAACGGAAATCTGAGAGTG
8771-PG R1 CACTCTCAGATTTCCGTTTGG
F-M基因座下游
Downstream of F-M		 8771-PG F2 TCCAGCTTAAGGCATCCACT 207 TCCAGCTTAAGGCATCCACTTGTTTCGGTTATGCAAGTGGATTAAGAATTGGATTGATTGTTGATGATGATATCACAACAAAAAAATTAGGCATGTACCATTATTCCAAGACTTCAGTTGGCCAAAGTCTCTTCCATGTGGAAATCTCTTCCTTGGTTCTAAGCTTCCTGATGCCCCCGTAAAACCCATCTGCCTCATTTCCCTCCT
8771-PG R2 AGGAGGGAAATGAGGCAGAT

Fig. 1

Phenotype of clingstone/freestone trait in peach A: 87-7-1, Clingstone; B: Zhongtao8, Freestone"

Fig. 2

Schematic diagram of the BAC library was screened by PCR"

Fig. 3

Agarose assays for PCR confirmed positive clones and the BAC clone plasmid A: M: DL 2000 DNA Marker; Lane 1-14: BAC clones 46-B-1-14 as template, and the positive control uses the clingstone with melting flesh peach Huangjinmi 3 as the template, while the negative control uses sterilized water as the template. B: M: TSINGKE 1.5 kb Marker, BAC: Plasmid extracted"

Fig. 4

Sequence alignment of PGs of BAC clone 46-B-10 and reference genome"

Fig. 5

Collinearity alignment of BAC clone and reference genome sequence The arrows indicate the location of upstream and downstream primers, and the dotted lines indicate the missing part"

Table 2

Sequence alignment of PGs of BAC clone 46-B-10 and reference genome"

基因ID
Gene ID		 参考基因组基因序列长度
Gene length of reference sequence (bp)		 BAC克隆基因序列长度
Gene length of BAC clone (bp)		 基因序列相似度
Similarity of gene sequences (%)		 编码氨基酸序列相似度
Similarity of coding amino acid sequences (%)
Prupe.4G261700 1519 1545 94.24 99.49
Prupe.4G261800 1771 1751 98.52 98.98
Prupe.4G261900 3262 3225 96.36 99.75
Prupe.4G262000 1516 1552 83.30 79.22
Prupe.4G262100 2380 / / /
Prupe.4G262200 2660 / / /
Prupe.4G262300 2544 / / /
Prupe.4G262400 3648 / / /
Prupe.4G262500 4553 4547 99.08 99.75

Fig. 6

PCR confirmed positive clones freestone/clingstone peach M: DL 2000 DNA Marker. 1-10: Freestone peach. Chunxue, Zaoyoutao, Zhongyou 22, Zhongtao 6, Xinzhongnan12-14, Xinzhongnan12-36, Xinzhongnan12-44, Xinzhongnan12-54, Xinzhongnan13-10, respectively; 11-20: Clingstone peach. Xinzhongnan12-55, Xinzhongnan12-59, Xinzhongnan12-60, Xinzhongnan13-4, Xinzhongnan13-14, 87-7-1, Zhongyou 8, Zhongyou 13, Zhongtao 5, Zhongyoutao 4, respectively; 21: The negative control uses sterilized water as the template"

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